The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to elucidate the mechanisms of action of the phorbol esters and their endogenous analog, the lipophilic second messenger sn-1,2- diacylglycerol. Protein kinase C (PKC) is the major receptor for these compounds, and our emphasis is correspondingly directed at his family of isozymes. In a collaborative effort, we seek to combine mutational analysis with computer modeling and chemical synthesis to probe ligand - PKC interactions. Our emerging insights have yielded synthetic ligands with nanomolar affinity and with unique selectivity between classes of diacylglycerol receptors. In particular, we now have ligands with enhanced selectivity for beta2-chimaerins compared to PKC. Using GFP-PKC fusion proteins, we are able to visualize PKC translocation in response to phorbol esters in living cells as a function of time. We find that different ligands cause different patterns of subcellular distribution of PKC delta. Differences in the hydrophobic domains of the ligands are important contributors to the different patterns of localization that they induce. Since different localization should confer access to different subsets of substrates, this mechanism may help account for the different biological responses to these ligands, which range from tumor promotion to inhibition of tumor promotion. Bryostatin 1 is a potent activator of PKC which paradoxically inhibits many biological responses to the phorbol esters and is currently in clinical trials as a cancer chemotherapeutic agent. Unlike typical phorbol esters, bryostatin 1 at high concentrations does not down regulate PKC delta, an anti-proliferative isoform, but rather protects it from down regulation. Although both phorbol ester binding domains (C1 domains) contribute equally to the down regulation of protein kinase C delta at low bryostatin concentrations, the first C1 domain plays the major role in the protection from down regulation. Although PKC is the major receptor for the phorbol esters, several novel families of proteins with C1 domains have now been identified which also recognize phorbol esters with high affinity. RasGRP, an activator of Ras, is of particular interest. RasGRP binds phorbol esters with similar affinity to PKC but with rather different lipid dependence. Variations in lipid composition can therefore provide discrimination between these two classes of phorbol ester receptors. The phorbol related diterpene resiniferatoxin acts as an ultrapotent analog of capsaicin and has permitted characterization of specific capsaicin (vanilloid) receptors. Its differential ability to desensitize vanilloid receptors makes it an attractive therapeutic candidate for treatment of chronic pain and other conditions in which vanilloid sensitive pathways are involved (e.g. urge incontinence). A vanilloid receptor VR1 has been cloned, and we find that it represents the major target for resiniferatoxin binding, despite rather different structure activity relations for ligand binding and for activation of its channel activity. Using GFP-VR1 constructs, we are able to visualize VR1 in intact cells and its response to ligands. Much of the receptor is located at internal sites, with important implications for its pharmacology. Using principles developed in the studies with PKC, ligands for VR1 with appreciably enhanced affinity compared to capsaicin have been developed. - capsaicin, vanilloid receptors, pain, phorbol ester, protein kinase C, tumor promotion, signal transduction, - Neither Human Subjects nor Human Tissues